Nucleoporin FG Domains Facilitate mRNP Remodeling at the Cytoplasmic Face of the Nuclear Pore Complex

Abstract

Directional export of messenger RNA (mRNA) protein particles (mRNPs) through nuclear pore complexes (NPCs) requires multiple factors. In Saccharomyces cerevisiae, the NPC proteins Nup159 and Nup42 are asymmetrically localized to the cytoplasmic face and have distinct functional domains: a phenylalanine-glycine (FG) repeat domain that docks mRNP transport receptors and domains that bind the DEAD-box ATPase Dbp5 and its activating cofactor Gle1, respectively. We speculated that the Nup42 and Nup159 FG domains play a role in positioning mRNPs for the terminal mRNP-remodeling steps carried out by Dbp5. Here we find that deletion (D) of both the Nup42 and Nup159 FG domains results in a cold-sensitive poly(A)+ mRNA export defect. The nup42DFG nup159DFG mutant also has synthetic lethal genetic interactions with dbp5 and gle1 mutants. RNA cross-linking experiments further indicate that the nup42DFG nup159DFG mutant has a reduced capacity for mRNP remodeling during export. To further analyze the role of these FG domains, we replaced the Nup159 or Nup42 FG domains with FG domains from other Nups. These FG "swaps" demonstrate that only certain FG domains are functional at the NPC cytoplasmic face. Strikingly, fusing the Nup42 FG domain to the carboxy-terminus of Gle1 bypasses the need for the endogenous Nup42 FG domain, highlighting the importance of proximal positioning for these factors. We conclude that the Nup42 and Nup159 FG domains target the mRNP to Gle1 and Dbp5 for mRNP remodeling at the NPC. Moreover, these results provide key evidence that character and context play a direct role in FG domain function and mRNA export. IN eukaryotes, messenger RNA (mRNA) export from the nucleus is an essential process that is highly regulated, with events sequentially coordinated to ensure proper RNA processing and cytoplasmic fate (reviewed in Moore 2005; MullerMcNicoll and Neugebauer 2013). In general, transcripts exit the nucleus through nuclear pore complexes (NPCs), large (60 MDa in yeast, 100 MDa in vertebrates) proteinaceous structures that provide an aqueous channel for ions, metabolites, proteins, and ribonuclear protein complexes (RNPs) to cross the nuclear envelope (reviewed in Wente and Rout 2010; Bilokapic and Schwartz 2012). For molecules .40 kDa, transport through the NPC is facilitated via transport receptors (TRs) that bind both the cargo and NPC proteins (nucleoporins, or Nups). For mRNA, binding to TRs is carefully coordinated to allow export only after the message is fully processed in the nucleus (reviewed in Muller-McNicoll and Neugebauer 2013).A key aspect of NPC function involves different NPC structures performing specific roles to ensure efficient transport. During NPC translocation, TRs interact with a class of Nups known as phenylalanine-glycine (FG) Nups (reviewed in Wente and Rout 2010). Each FG Nup has an unstructured domain enriched with FG, glycine-leucine-phenylalanineglycine (GLFG), or phenylalanine-any amino acid-phenylalanine-glycine (FxFG) repeats, which are flanked by characteristic...

Integrative structure and functional anatomy of a nuclear pore complex

“…6D; Extended Data Fig. 9C), consistent with the known role of FxFG/FG-type repeats in docking exporting RNAs 31 . In contrast, the GLFG-type repeats are enriched in regions adjacent to the inner ring and near the cytoplasmic entrance to the central channel.…”

Nup42 and IP6 coordinate Gle1 stimulation of Dbp5/DDX19B for mRNA export in yeast and human cells

“…Our prior studies revealed a role for the FG domain in recruiting the mRNP to be in proximity to Gle1 and Dbp5 for remodeling 28 , via the Nup42 FG domain interacting with Mex67-Mtr2 (Figure 1B) 29 . In fact, a fusion between Gle1 and the FG domain of Nup42 (gle1-FG nup42 ) bypasses the requirement for Nup42 in some genetic contexts 28 . The Nup42 CTD is also required for specific functions.…”

“…Nup42/hNup42 mediates the export of heat shock transcripts 22,30 , and deletion of NUP42 together with IPK1 , the kinase that produces IP 6 , results in a temperature sensitive mRNA export defect 31 . Nup42 CTD expression is sufficient for the function in heat shock mRNA export 32 and rescues nup42△ ipk1△ temperature sensitivity 31 ; whereas, expression of the gle1-FG nup42 fusion does not restore these defects in nup42△ mutants 28 . Since proper Gle1 and Dbp5 function are likewise required for heat shock mRNA export 30,33 , and IP 6 is required for normal Gle1 regulation of Dbp5, we propose that the Nup42 CTD might also impact their interaction.…”

Comparative interactomics provides evidence for functional specialization of the nuclear pore complex

“…9,36 This asymmetry is required for aspects of NPC function, especially mRNA export, which relies on the cytoplasmically biased Nup82 complex for messenger ribonucleoproteins (mRNPs) remodeling before release into the cytoplasm for translation by ribosomes. 15,37 There are significant differences between opisthokont and trypanosome FG-motif domain sequences, making it near impossible to identify orthologs of opisthokont FG-Nups in trypanosomes by in silico means alone. However, through affinity capture, we were able to identify orthologs of FG-Nups, based on the core scaffold structure with which they associate.…”

“…40 A major source of Nup asymmetry in opsithokonts is the exclusively cytoplasmic ScNup82/HsNup88 subcomplex, tethering specialized FG-Nups that provide the interaction platform for factors critical for mRNA export. 15,37 However, TbNup76, the presumed trypanosome ortholog of ScNup82, is located on both the cytoplasmic and nucleoplasmic faces of the trypanosome NPC. 5 Herein lies another cautionary tale of making sure that one does not assume that, just because proteins are orthologous, they function in the same way in organisms that are evolutionarily distant.…”